A tumor-associated antigen present on chronic lymphocytic leukemia (CLL) cells has been identified with rabbit xenoantisera prepared against intact CLL cells. The resultant antisera were extensively absorbed with AB erythrocytes, HLA-matched peripheral blood lymphocytes (PBL) and enriched B cells carrying a common alloantigen. Using the 51Cr-microcytotoxicity assay and the indirect fluorescent antibody technique, the CLL-associated antigen could not be detected on normal or T or B lymphocytes, cultured lymphoblastoid cell lines, mitogen-stimulated PBL or thymus, spleen, bone marrow or lymph node cells. In addition, the tumor antigen was not expressed on malignant cells from other types of leukemia (AML, CML, ALL) but was present on cells from patients with hairy cell leukemia. The tumor antigen was isolated from CLL plasma membranes by a combination of immunoaffinity and ion-exchange chromatography. Using SDS-PAGE, the tumor antigen was shown to be composed of a single polypeptide with an estimated molecular weight of 69,000.